Protein Immunoprecipitation from a Yeast Cell Lysate

  1. Grow desired volume of cultures to OD600 0.5 (~1E7 cells/ml) using selective media.
  2. Harvest cells by centrigugation.
  3. Lyse cells as follows:
    1. Harvest 2.0 ml cells in a micro-centrifuge by spinning full speed for 3 minutes.
    2. Decant and discard supernatant.
    3. Add 200 ul of SUME buffer + protease inhibitors.
    4. Add 100 ul of 0.5 mm Acid Washed Glass Beads.
    5. Vortex in multivortexer or by hand, 3 X 1 min full speed on our current machine.
    6. Remove the lysate from the beads with a blue pipette tip to a new 1.5 ml eppendorf tube.
    7. Spin 5 minutes to clarify. Remove supernatant to a new 1.5 ml eppendorf tube.
  1. Add 1.0 ml IP buffer to each sample.
  2. Add 10 ul antibody to each sample, and incubate at 4oC overnight.
  3. Add 100 ml of 10% ProteinA-Sepharose to each sample, and incubate at room temperature for 2 hours.
  4. Spin samples at 1000 rpm in microcentriguge for 15 seconds, then carefully remove lysate and save in a new 1.5 ml eppendorf tube.
  5. Wash ProteinA-Sepharose beads once with IP buffer and twice with wash buffer by resuspending beads in buffer, incubating 1 minute and microcentrifuging at 1000rpm for 15 seconds. Apsirate the supernatant from the beads.
  6. Add 50 ul SUME buffer + 0.005% bromophenol blue to ProteinA-Sepharose beads and incubate at 65oC, or other desired temperature, for 10 minutes.
  7. Load 10-30 ul samples onto a SDS-PAGE gel.
  8. After running gel, transfer proteins to nitrocellulose and perform a western.

Note that protease inhibitors are added from stocks to give 50 fold dilution.
Example: 20 ul of stock per 1ml buffer.
Use IMMEDIATELY after adding protease inhibitors.
SUME Buffer
(1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA)
To make 100ml:
  • 1.0ml 1M MOPS, pH6.8 stock solution
  • 1.0g SDS, or 10ml 10% SDS stock solution
  • 48.05g Urea
  • 2.0ml 0.5M EDTA stock solution
This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.

Immunoprecipitation (IP) Buffer
(15mM Na2HPO4, mw 142; 150mM NaCl, mw 58; 2% Triton X-100, 0.1% SDS, 0.5% DOC, 10mM EDTA, 0.02% NaN3)
To make 100ml:
  • 0.213g Na2HPO4
  • 0.87g NaCl
  • 2.0ml TritonX-100
  • 0.1g SDS or 1ml 10% SDS stock solution
  • 0.5g deoxycholate
  • 2.0ml 0.5M EDTA stock solution
  • 0.2ml 10% NaN3 stock solution (optional)
Final pH should be 7.5. Sterilize through a sterile filter if azide (NaN3) is not added. Wear a mask while handling the SDS. Requires Heat to get SDS into solution.

Wash Buffer
(50mM NaCl, mw58; 10mM TRIS, mw 121; 0.02% NaN3)
To make 100ml:
  • 0.213g Na2HPO4
  • 0.29g NaCl
  • .12g Tris
  • 0.2ml 10% NaN3 stock solution (optional)
Final pH should be 7.5. Sterilize through a sterile filter if azide (NaN3) is not added.

50X Stock Protease Inhibitors (store at -20oC)

PMSF (87 mg/ml)
([500mM] phenylmethylsulfonyl fluoride)
To make 30 ml:  Dissolve 2.61g PMSF in DMSO to equal a final volume of 30 ml.

LEUPEPTIN AND PEPSTATIN (5 mg/ml)
To make 10 ml:  Dissolve 50mg LEUPEPTIN or PEPSTATIN in DMSO to equal a final volume of 10 ml.

TPCK (5 mg/ml)
(tosylphenylalanine chloromethyl ketone)
To make 10 ml:  Dissolve 50mg TPCK in DMSO to equal a final volume of 10 ml.

* Note: The EDTA in the SUME buffer is also a protease inhibitor.


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